Method for modulating steroidogenic activity

ABSTRACT

The present invention relates generally to a method for modulating steroidogenic activity and a composition useful for same. The present invention further relates to a composition comprising a steroidogenic modulator useful for modulating physiological processes mediated by the androgen receptor or an active form thereof or complex comprising same and/or for modulating physiological processes mediated by estrogen receptors. The composition of the present invention preferably comprises an extract of herbs or botanical or horticultural equivalents of the herbs or chemical or functional equivalents of one or more components of the herbal extract thereof.

This application is a Continuation of co-pending application Ser. No.09/793,772, filed on Feb. 27, 2001, and for which priority is claimedunder 35 U.S.C. § 120; and this application claims priority of U.S.Provisional Application No. 60/185,757 filed on Feb. 29, 2000 under 35U.S.C. § 119(e); the entire contents of all are hereby incorporated byreference.

FIELD OF THE INVENTION

The present invention relates generally to a method for modulatingsteroidogenic activity and a composition useful for same. The presentinvention further relates to a composition comprising a steroidogenicmodulator useful for modulating physiological processes mediated by theestrogenic or androgenic receptor or an active form thereof or complexcomprising same and/or for modulating physiological processes mediatedby the estrogen and androgen receptors. The composition of the presentinvention preferably comprises an extract of herbs or botanical orhorticultural equivalents of the herbs or chemical or functionalequivalents of one or more components of the herbal extract.

BACKGROUND OF THE INVENTION

Bibliographic details of the publications referred to by author in thisspecification are collected at the end of the description.

Herbal formulations comprising extracts of more than one herbal planthave been used for centuries in Traditional Chinese Medicine (TCM).There is now an increasing acceptance of their value and therapeuticefficacy in Western medicine. TCM has its own unique and philosophicaltheory in etiology, pathology, diagnosis, pharmacology and therapeutics.Many concepts surrounding TCM have particular relevance to Westernmedicine such as viewing parts of the body as an organic whole,considering inter-relations and influences between organs and beingaware of relevant adaptation of the human body to the naturalenvironment.

Despite the effectiveness of many herbal formulations in the treatmentof a range of conditions, little is known about how the formulationswork. Information on the mechanisms of action for herbal formulationswould permit the rational design of particular compositions or chemicalsynthetic production of one or more components of the compositions aswell as ensuring that the appropriate composition is selected for aparticular ailment or condition.

Androgens are one of a group of steroid hormones which includetestosterone and dihydrotestosterone (DHT). The androgens stimulate thedevelopment of male sex organs and male secondary sexual characteristicssuch as beard growth, deepening of the voice and muscle development. Theprincipal source of these hormones is the testis but they are alsosecreted in small amounts from the adrenal cortex.

Androgens act through an X-linked androgen receptor (AR) to regulateandrogen-responsive genes. This in turn leads to a cascade of metabolicevents which manifest as androgenic effects including male sexualdevelopment in the fetus, secondary sexual development and spermproduction at puberty, anabolic processes including muscle growth andbone density, male sex drive (i.e. libido), hair growth, skin condition,and physical stamina in adults (Wilson, 1992).

Naturally occurring and synthetic androgens are used in replacementtherapy such as to treat delayed puberty in boys, hypogonadal men,impotence and as anabolic agents and in the treatment of cancer.However, only limited number of natural and synthetic androgens areknown. As stated above, testosterone and DHT are examples of naturalandrogens. Miborelone and mesterolone are examples of syntheticandrogens. The chemical structure common to steroidal hormones,including androgens, is the 1,3-cyclopentanophrenanthrene ring system.

Androgens in the cell bind to the ligand-binding domain (LBD) of theandrogen receptor. Upon ligand binding, the androgen receptor whichcomprises the transactivation domain (TAD), DNA-binding domain (DBD) andthe ligand bound LBD adopts a transactivational conformation andtranslocates to the nucleus where it binds specifically to theandrogen-responsive element (ARE) of the androgen-regulated gene.Following the recruitment of DNA polymerase and co-activators to formthe quaternary transcription complex, the gene downstream of the ARE isexpressed. Hence, the prerequisite of androgen receptor activity is thespecific binding of a ligand into the hydrophobic core of the androgenreceptor LBD.

As stated above, there are many conditions associated with low androgenlevels, hypofunction of the androgen receptor (Yong, 1994; Tut, 1997;Lim, 1997; Yong, 1998; Wang, 1998, Ghadessy, 1999; Ong, 1999; Dowsing,1999), declining androgen action associated with aging and otherconditions.

There is a need to identify naturally occurring compounds and materialsgenerally from non-mammalian sources, which interact with or activatethe androgen receptor and/or the androgen-androgen receptor complexleading to transcription of an androgen-responsive gene. There is also aneed to identify estrogenic modulating agents, as well as agentsmodulating the effects of other members of the steroid/nuclear receptorsuperfamily of proteins. The identification of such compounds andmolecules is needed for the development of therapeutic compositionsand/or nutraceutic applications.

In work leading up to the present invention, the inventors sought anedible plant extract with steroidogenic properties.

Eucommia Ulmoides OLIVER (Du-Zhong) is a large deciduous tree whichoriginated in China. The bark of the tree (commonly referred to asCortex eucommiae) has been used for natural medicine since ancient times(Wei, 1955; Li, 1987).

Decoctions of E. Ulmoides (EU) bark have been used for, amongst otherthings, the relief of back pain, to increase strength, to make bones andmuscle strong, to increase recovery from fatigue, to increase ability toremember and to induce an anti-aging effect. Mechanical training and theuse of EU leaf extracts co-operatively can increase the ability of ratsto avoid lactate accumulation in skeletal muscle and the administrationof the EU leaf extract along with light intensity training enhances theability of a muscle to resist fatigue (Li, 1996b). EU leaves containcompounds similar to the bark and are reported to have similarpharmacological effects. Since irridoid monoglycosides, such asgeniposidic acid and aucubin in EU can stimulate collagen synthesis inaged model rats (Li, 1991a), it is thought that the active compound isactually geniposidic acid or aucubin.

Crude extracts of Tochu tea, an aqueous extract of EU leaves, have asuppressing effect on the induction of chromosome aberrations in CHOcells and mice. Out of 17 Tochu tea components, five irridoids(geniposidic acid, geniposide, asperulosidic acid, deacetylasperulosidic acid and asperuloside) and three phenols (pyrogallo,protocatechuic acid and p-trans-coumaric acid) were found to haveanti-clastogenic activity (protective effect against chromosomalaberrations). Since the anti-clastogenic irridoids had analpha-unsaturated carbonyl group, this structure was considered to playan important role in the anti-clastogenicity (Nakamura, 1997).

Ingestion of EU bark and leaves, and/or their extracts, cause no knownside effects.

In accordance with the present invention, the inventors have determinedthat certain extracts of EU exhibit steroidogenic activity. Theidentification of the activity in EU extracts permits the rationaldesign of therapeutic protocols and compositions useful in amelioratingthe symptoms of disease conditions. It also permits the production ofthe active agents in the extracts in purified or chemical syntheticform.

SUMMARY OF THE INVENTION

In the context of the present invention, a composition “consistingessentially of” recited ingredients will elicit physiological conditionsand responses mediated by estrogen or estrogen receptor or androgens orandrogen receptors, preferably in a synergistic manner compared to theresponse obtained using estrogen or androgen alone as the elicitingcompound. As a preferred instance of synergistic effect, a basal degreeof activity of steroid alone or of the composition alone might betwo-fold activation of the steroid receptor activity. Synergistic actionis observed when greater than four-fold activation is observed,preferably when greater than six-fold activation is observed, morepreferably when greater than eight-fold activation is observed, evenmore preferably when greater than 10-fold activation is observed whenthe steroid and the composition of the invention are applied together inthe assay.

One aspect of the present invention is a method of extracting activesteroidogenic compounds from EU plants. The steps comprise maceratingthe tissues of the EU plant, extracting the active compounds withsteroidogenic activities with a solvent system, separating the liquidfrom the solid phase and adding water to precipitate the undesiredcompounds that may cause side-effects and/or reduce efficacy of the mainactive compounds with steroidogenic activities. As tissues of the EUplant, all tissues can be used; bark or leaves are preferred, and barkis most preferred.

According to this invention, the term ethanolic EU extract refers to EUextract using a solvent system consisting only of ethanol. The termhydroethanolic EU extract refers to the EU extract using a solventsystem comprising ethanol and 20% water, in which the water componentcould be added before or after the extraction process. For the purposeof the examples, the extract was dried, weighed and resuspended inethanol at a known concentration.

Another aspect of the present invention contemplates a method ofmodulating a steroidogenic-mediated physiological condition in asubject, said method comprising administering to said subject aneffective amount of a formulation comprising an extract of EU orbotanical or horticultural equivalents of EU or chemical or functionalequivalents of the extract or a purified, or chemically synthesized formof one or more components of the extract. Another aspect of the presentinvention is directed to a composition comprising a part of EU or abotanical or horticultural equivalent of EU or an extract thereof orchemical or functional equivalents of the extract or a purified, orchemical synthetic form of one or more components of the extract whereinsaid composition is effective in modulating a steroidogenic-mediatedcondition in a subject.

Another aspect of the invention is an article of manufacture thatcomprises an extract of the invention, or a purified or chemicallysynthesized molecule that is a component of the extract, that hassteroidogenic activity, preferably synergistic activity together with asteroid compound (especially with an androgen or estrogen compound). Insuch an article of maufacture, the extract or purified or synthesizedcomponent is packaged together with written materials that provideinstructions or describe or urge use of the extract or purified orsynthesized component to modulate, and especially to enhance, aphysiological condition or response mediated by a steroid, especially acondition or response mediated by an androgen receptor or by an estrogenreceptor.

Yet another aspect of the present invention is directed to a purified orchemical synthetic molecule form of EU, or a botanical or horticulturalequivalent thereof, or an extract thereof which molecule is capable ofmodulating a steroidogenic-mediated condition.

Still another aspect of the present invention provides for the use of EUor a botanical or horticultural equivalent thereof or an extract thereofor a chemical or functional equivalent of the extract or a purified orchemical synthetic form of one or more components of the extract in themanufacture of a medicament for the treatment of steroidogenic-mediatedconditions.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A to E are graphical representations showing the dose-dependentresponses of five treatments: (a) androgenic activity of an ethanolic EUextract; (b) androgenic activity of an hydroethanolic EU extract; (c)testosterone-potentiating activity of a fixed dose of ethanolic EUextract (50 ng dry weight/ml treatment medium) with increasing doses oftestosterone; (d) DHT-potentiating activity of a fixed dose of ethanolicEU extract (50 ng dry weight/ml treatment medium) with increasing dosesof DHT; (e) DHT-potentiating activity of increasing doses of theethanolic EU extract with a fixed dose of DHT (1 nM).

Androgen-induced transactivation activity was used as a measure ofandrogenic activity and was measured with an androgen-regulated reportergene, ARE-Tata-Luc, and expressed as fold increase in luciferaseactivity compared with cells not exposed to androgen. Each data point isthe mean +(SE) of triplicate samples.

HeLa cells transiently expressing androgen receptor were exposed toincreasing doses of ethanolic EU extract (FIG. 1 a). Similarly, androgenreceptor expressed in HeLa cells were also exposed to increasing dosesof an ethanolic EU extract precipitated with 20% deionized water(hydroethanolic EU extract) (FIG. 1 b). Treatments were also carried outusing increasing doses of testosterone with an ethanolic EU extract at afixed concentration of 50 ng/ml (FIG. 1 c), increasing doses of DHT withan ethanolic EU extract at a fixed concentration of 50 ng/ml (FIG. 1 d)and 1 nM DHT with different doses of an ethanolic EU extract (0 ng/ml to50 ng/ml) (FIG. 1 e).

FIG. 2 is a graphical representation showing radioligand displacementassay of an ethanolic EU extract. COS-7 cells were transfected with DNAencoding AR and then exposed to 3 nM of tritiated testosterone and theindicated amounts of DHT (nM), cortisol (nM) or ethanolic EU extract(concentration 1=50ng/ml) for 2 hours at 37 ° C. The treated cells wereharvested and the amount of tritiated testosterone bound to AR ismeasured by scintillation counting. Specific binding is expressed aspercent tritium bound to AR, where 100% is the amount of specifictritiated-testosterone bound in the absence of competing cold ligandminus background (non-specific binding to substrate and proteins). Eachdata point, the mean of quadruplicates, represents the amount ofradiolabelled testosterone specifically bound on exposure to indicateddoses of DHT, cortisol or ethanolic EU extract.

FIG. 3 is a graphical representation of estrogenic effect of EU. Helacells were transfected with DNA encoding human estrogen receptor and theestrogenic effect of an ethanolic EU extract measured with MMTV-ERE-LUCreporter gene. Control cells were not exposed to the ethanolic EUextract while other cells were exposed to indicated doses of theethanolic EU extract. Estrogenic activity is expressed as fold increasein reporter gene activity compared to control. Data are mean ±SE oftriplicate samples.

FIG. 4 is a graphical representation of the synergistic effect of anethanolic EU extract on estradiol action. Hela cells were transfectedwith DNA encoding human estrogen receptor and the estrogenic effect wasmeasured with a MMTV-ERE-LUC reporter gene. Cells were exposed toincreasing doses of estradiol, with or without 50 ng/mL of the ethanolicEU extract, as indicated. Control cells were not exposed to theethanolic EU extract or estradiol. Estrogenic activity is expressed asfold increase in reporter gene activity compared to control. Data aremean ±SE of triplicate samples.

FIG. 5 is a graphical representation of the effect of increasing dosesof an ethanolic EU extract on estradiol action. Hela cells weretransfected with DNA encoding human estrogen receptor and the estrogeniceffect was measured with a MMTV-ERE-LUC reporter gene. Cells wereexposed to 0. nM estradiol and increasing doses of the ethanolic EUextract as indicated. Control cells were not exposed to the ethanolic EUextract. Estrogenic activity is expressed as fold increase in reportergene activity compared to control. Data are mean ±SE of triplicatesamples.

FIGS. 6A to 6C are graphical representations of androgenic studies inanimal models demonstrating the synergistic effect of an ethanolic EUextract. The growth of the ventral prostate gland, an androgen-dependenttissue, in immature Wistar rats was measured. Administration oftestosterone by intramuscular injection alone resulted in dose-dependentincrements in prostatic weight (FIG. 6A). Oral dosing of the animalswith the ethanolic EU extract alone, at doses from 1 /mg to 10 /mg, alsoresulted in dose-dependent increments in prostatic weight (FIG. 6B).Treatment of animals by intramuscular injection of a high dose oftestosterone (5 ug) and also orally feeding 50 /mg of the ethanolic EUextract indicated a highly significant synergistic effect whereby theethanolic EU extract was able to increase prostatic growth to above thatobserved with the high dose of testosterone alone (FIG. 6C).

FIG. 7 a is a graphical representation of the solid phase separation ofandrogenic activity of compounds in an ethanolic EU extract. A 50 mlDiol matrix was loaded into a glass column and an ethanolic EU extract(50 mls) was dry packed on top of the Diol matrix. The ethanolic EUextract was then sequentially eluted into individual fractions using 150ml of each of the following solvents of increasing polarity in thefollowing order: hexane 100%, hexane:dichloromethane (DCM) (1:1), DCM100% and methanol (MeOH) 100%. The fractions were dried down in a rotaryevaporator at 37 ° C. and resuspended in equivalent amounts of ethanolfor assay of androgenic activity as in FIG. 1. All experiments werepreformed in the presence of 1 nM of the synthetic androgen, miborelone(MB) and data are expressed with MB activity as 100%.

FIG. 7 b is a graphical representation of the solid phase separation ofandrogenic activity of compounds in an ethanolic EU extract using moredefined solvents. Experiments were conducted as in FIG. 7 a except thatthe following solvents were used DCM, ethyl acetate and ethanol (EtOH).The column was eluted first with 150 ml of DCM, then followed by 150 mlof ethyl acetate and finally with 300 ml of EtOH. The EtOH eluate wasindividually collected into two separate, sequential fractions: EtOH Iand EtOH II. All experiments were performed in the presence of 10 nM ofthe physiological androgen, dihydrotestosterone (DHT).

FIG. 8. Fractionation of an ethanolic EU extract with flashchromatography using Sephadex LH-20 followed by thin layerchromatography (TLC) silica gel 60 matrix in conjunction withbio-characterisation using AR gene expression. FIG. 8 a shows that ann-butanolic fraction (F1) from a Sephadex LH-20 separation of theethanolic EU extract was further resolved into distinct bands (B0, B0-1,B 1, B2, B3, B4, B4-end) which can be visualized with either ultraviolet(UV) illumination or with a chemical reagent (vanillin-sulphuric acid).Upon bioassays with the AR, the agonistic activity of the ethanolic EUextract can be separated into phytocompounds with widely/differinghydrophilicity by TLC (FIG. 8 b). TLC band B0-1 (morestrongly-interacting with silica gel) can activate the AR in anagonistic manner as indicated in FIG. 8 b, at relative concentrations of1 concentration factor (cf) and 3 cf. Similarly, band B4-end alsodemonstrates agonistic bioactivity albeit only more markedly at a higherconcentration of 3 cf.

FIG. 9. Fractionation and bioactivity characterisation of an ethanolicEU extract with high performance liquid chromatography (HPLC) using areverse phase C-18 semi-preparative column in conjunction withdownstream AR bioassays. The HPLC column was eluted with an acetonitrilegradient mobile phase. (A chromatogram is shown as FIG. 10.) F1 is thefraction that elutes between 40 min to 60 min time interval. F2 is thefraction eluting between 60 min to 80 min while F3 shows bioactivity offraction eluting between 80 min to 120 min. The Figure shows biologicalactivity of the fractions. The black bar indicates AR activity in thepresence of the androgen dihydrotestosterone (DHT). F1 demonstrates bothandrogen-like (agonist) activity on its own (fraction alone) and alsoandrogen-boosting activity in the presence of DHT (fraction+DHT). Incontrast, F2 and F3 show only minimal agonist activity (fraction alone)but demonstrate strong androgen-boosting activity in the presence of DHT(fraction+DHT).

FIG. 10. HPLC Chromatogram. Elution profiles of three runs of thereverse-phase HPLC chromatography performed as in Example 9 aresuperimposed. Fractions F1, F2 and F3 are indicated between verticalarrows. The acetonitrile gradient is shown v. time.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is predicated in part on the identification ofsteroidogenic-modulating properties in a formulation comprising parts ofEU or an extract thereof. Accordingly, one aspect of the presentinvention is a method of extracting active steroidogenic compounds fromEU plants. EU plant parts are macerated prior to the actual extractionprocess. Fresh EU plant parts or preferably dried plant parts aremacerated using any of the known process such as chopping into smallpieces, grinding into powder or breaking up into fine particles using ahigh speed blender. Although active compounds with steroidogenicactivities can be extracted from different parts of the EU plant, thepreferred part of the EU plant for extraction is the bark.

A soaking method is one of the several methods of extracting the activecompounds from the EU plant. In this method, the macerated plant issoaked with a solvent system and left for a period of time to allow theactive compounds to dissolve into the solvent system. To enhance thediffusion of the active compounds into the solvent system, the mixturecan be mechanically agitated and/or heated to a pre-determinedtemperature. The mechanical agitation methods include but are notlimited to the following: shaking, vortexing, swirling, stirring andultrasonicating.

After a sufficient period of time for the diffusion of the activecompounds into the solvent system, the liquid is separated from thesolid by any one of the well-known techniques such as filtration andcentrifugation.

The solvent system can comprise any of the well-known systems such as anorganic solvent or a combination of organic solvents. Preferably, theorganic solvent is alcohol and more preferably, ethanol. Other organicsolvents such as hexane, dichloromethane, ethyl acetate will also beeffective. Water can be part of the solvent system. The percentage ofwater can range from 0% to 100%. If water is not part of the initialsolvent system, water can be added to the liquid phase during any partof the soaking period. It is also possible to add water to the liquidphase after separation from the solid phase. One of the effects of theaddition of water is to cause precipitation of non-steroidogeniccompounds that may have adverse side-effects. Water can be added up to20% by volume.

Another method of extracting active compounds from the macerated EUplant is to percolate the macerated EU plant with a continuouslyrefluxed solvent system such as the soxlet-type method for extraction.After completion of the extraction process, the liquid containing theactive compounds may be mixed with water (up to 20% by volume) toprecipitate the undesired compounds. The precipitate can be separatedfrom the liquid containing the active compounds with steroidogenicactivity using any one of the well known separation methods such asfiltration and/or centrifugation.

The solvents in the liquid phase containing the active compounds withsteroidogenic effects can be evaporated off using any of the well knowndrying methods including but not limited to, rotary evaporation,speed-vacuum centrifugation or open-top drying. The dried extract isthen suitable for use or storage. If desirable, the dried extract can beresuspended in suitable solvents prior to use.

For further purification, the extract, either dried and then redissolvedin an appropriate solvent, preferably water or an alcohol, can beapplied to a reverse phase chromatography column and the steroidogeniccompounds can be eluted with a mobile phase comprising water andacetonitrile. Alternatively, or in combination with the reverse phasepurification, the extract can be applied to a chromatography matrixcomprising dextran crosslinked by an alkyl ether and the steroidogeniccompounds can be eluted with an alcohol, preferably a C₁-C₆ alkylalcohol, more preferably an n-alkyl alcohol.

Accordingly, another aspect of the present invention contemplates amethod of modulating a steroid-mediated physiological condition in asubject, said method comprising administering to said subject aneffective amount of a formulation comprising an extract of EU orbotanical or horticultural equivalents of EU or chemical or functionalequivalents of the extract or a purified or chemical synthetic form ofone or more components of the extract.

A “steroid-mediated physiological condition” includes anandrogen-mediated physiological condition, an estrogenic-mediatedphysiological condition and/or other physiological condition mediatedthrough the steroid/nuclear receptor family of proteins. A“steroidogenic agent” includes an androgen, an estrogen and/or any otherligand interacting with the steroid/nuclear receptor family of proteins.Estrogenic agents are useful in hormonal therapy in hypoestrogenicstates such as but not limited to menopause, osteoporosis andcardiovascular disease. An “estrogen-mediated physiological condition”also includes and encompasses conditions mediated via an estrogenicreceptor or active or complex forms thereof.

Reference herein to an “androgen-mediated physiological condition”includes reference to the induction of physiological processes mediatedvia the androgenic receptor. These physiological processes includebiological, endocrinological and other bodily processes which areinduced, stimulated, enhanced or otherwise facilitated by the androgenreceptor or androgen receptor complexes and/or its activated forms whichare responsive to natural or synthetic androgens or other compoundswhich have androgenic properties such as being able to activate theandrogen receptor.

The ability to activate or otherwise modulate the androgen and/orestrogen receptor may be tested in vitro or in vivo. Reference to invivo includes the practice of the present invention in humans, primates,livestock animals (e.g. sheep, cows, pigs, goats, horses, donkeys),laboratory test animals (e.g. rabbits, mice, rats, guinea pigs),companion animals (e.g. dogs, cats) and captive wild animals.

For convenience, a “steroid receptor” encompasses the androgen receptorand/or estrogen receptor and/or other receptor belonging to thesteroid/nuclear receptor family of proteins. A “steroidogen” encompassesan androgen, an estrogen and/or any other ligand interacting with thesteroid/nuclear receptor family of proteins.

The term “modulate” and its variations including “modulating” and“modulates” includes the up-regulation and down-regulation ofsteroidogenic receptor activity or the activity ofsteroidogen-steroidogen receptor complex activity. This may beconveniently determined at the level of up-regulating or down-regulatingtarget gene expression following modulation of steroidogen receptoractivity.

The present invention is particularly directed to a formulationcomprising EU or parts thereof or an extract thereof or botanical orhorticultural equivalents of EU or chemical or functional equivalents ofthe extract from EU extract or purified or chemical synthetic forms ofone or more components in EU or an extract thereof.

Reference herein to “Eucommia Ulmoides” or “E. Ulmoides”, or “Du Zhong”,or “EU” includes reference to botanical and horticultural equivalentsthereof. Botanical and horticultural equivalents includes herbs andother plants related at the genetic, biochemical, or medicinal level toEU. For example, a medicinally functional equivalent plant may beindigenous to another country. Such a plant is encompassed by thepresent invention. Botanical equivalents of EU are described by Gu etal. (Z. M. Gu et al., Chung Kuo Chung Yao Tsa Chih 14:714-7171 (1989))and encompass plants that are used among different ethnic groups assubstitutes for cortex eucommiae. Their study and identification ofsamples of all original plants and materia medica from all locationsproducing Du Zhong identified 48 species of ethnic substitutes for DuZhong from 17 genera of 10 families:

-   -   1. Lauraceae Litsea 1 species    -   2. Anacardiaceae Rhus 1 species    -   3. Rosaceae Potentilla 1 species    -   4. Actinidiaceae Saurauia 1 species    -   5. Malvaceae Urena 1 species    -   6. Bignoniaceae Catalpa 1 species    -   7. Apocynaceae        -   (1) Alstonia 1 species        -   (2) Parameria 1 species        -   (3) Beaumontia 1 species        -   (4) Chonemorpha 2 species        -   (5) Trachelospermum 1 species        -   (6) Ichnocarpus 1 species        -   (7) Parabarium 4 species    -   8. Asclepiadaceae        -   (1) Gymnema 2 species        -   (2) Marsdenia 1 species    -   9. Araliaceae Hedera 1 species    -   10. Celastraceae Euonymus 27 species

Horticultural equivalents of of EU include callus, meristem or tissuesor cells maintained in in vitro culture.

Reference to the Du Zhong herb also encompasses natural and artificiallycreated variants of EU. An artificially created variant includes avariant made by selective breeding or by genetic manipulation. A part ofEU includes the bark, leaf, stem, root, flower, seed or otherreproductive or vegetative portion of the plant or a combination of twoor more of these portions.

The term “formulation” includes an extract of EU or parts thereof inliquid, solid or aerosol or vapour form. In a preferred embodiment, theformulation comprises an ethanolic or aqueous extract of EU.

Herbal extraction techniques were designed to maintain maximal levels ofactive components. The steps comprise macerating the preferred bark ofthe EU plant, extracting the active compounds with steroidogenicactivities with a solvent system, separating the liquid from the solidphase and optionally adding water of up to 20% v/v to precipitate theundesired compounds that may cause side-effects and/or reduce efficacyof the main active compounds with steroidogenic activities. The solventsystem is preferably an C₁-C₆ alcohol, which can also include water inan amount up to 20% v/v. The separation of the solvent extract frominsoluble solids can be performed by any method typical in the art.Filtration is preferred, but centrifugation can also be efficientlyemployed.

The term “ethanolic EU extract” as used herein refers to EU extractusing a solvent system consisting only of ethanol. A “hydroethanolic EUextract” refers to the EU extract using a solvent system comprisingethanol and 20% water, in which the water component could be addedbefore or after the extraction process.

As defined herein, the EU extract is considered to exhibit steroidogenactivity. More particularly, the extract itself or one or morecomponents therein are considered herein to be “androgen modulators” or“estrogen modulators” (i.e. steroidogen modulators) in that the extractor its components are capable of modulating the activity of the androgenreceptor or a complex comprising same and/or the activity of theestrogen receptor or a complex, etc. The steroidogen modulators of thepresent invention are isolatable or obtainable from EU are defined asbeing “phyto-androgens” or “phyto-estrogens” (i.e. phyto-steroidogens)due to their botanical origin. Reference to an “androgen” in the term“phyto-androgen” is not to imply any limitation as to the structure ofthe phyto-androgen and the term extends to any component of EU or anyextract of EU or any component or extract from a botanical orhorticultural relative of EU which is capable of modulatingandrogen-receptor activity. Androgen receptor activity is convenientlymeasured in vitro or in cell culture by assays of transactivation and/ordownstream target gene expression. “Downstream target genes” whoseexpression is regulated by androgen receptor activation are known in theart. Transactivation assays, in which androgen or other steroid receptorbinding sites are coupled to reporter genes and then these constructsare placed into cells either in vitro or in vivo, are well-known in theart. Androgen receptor activity can also be assessed in vivo bymeasurement of physiological or anatomical parameters, e.g. developmentof male sexual organs, increase in muscle mass or spermatogenesis, knownin the art to be regulated by androgen activity. Accordingly, the term“androgen modulator” includes a formulation or composition or extract ofEU or a part of EU or a purified or chemical synthetic form of acomponent of the extract or part of EU or its botanical or horticulturalequivalent. Similar comments, in relation to development of femininecharacteristics, including regulation of serum levels of hormonesrelated to the estrus cycle, apply in relation to “phyto-estrogen”.

Reference herein to the “androgen receptor” or “estrogen receptor”includes reference to the naturally occurring receptor or itsrecombinant forms as well as splice variants or other genetic variantsincluding polymorphic variants. Furthermore, the term encompassescomplexes comprising the receptor and other molecules (e.g. androgen orestrogen) as well as the receptor's monomeric, dimeric, trimeric ormultimeric forms including homodimeric, homotrimeric, homomultimeric,heterodimeric, heterotrimer and heteromultimeric forms. The receptor maybe membrane associated or it may have translocated to the nucleus or beassociated with chromosomal DNA.

The androgen receptor when activated by androgens, includingphyto-androgens, has the intrinsic ability to bind to specific DNAsequences. Following binding, the transcriptional activity of a targetgene (i.e. a gene associated with the specific DNA sequence) ismodulated as a function of the ligand bound to the receptor. Functionalactivity of androgen receptors can be measured with reporter gene(s),such as firefly luciferase, coupled to steroid response elements, thatare co-expressed with the androgen receptor (Quigley, 1995).

Processes capable of being modulated by an androgen or via an androgenreceptor, in accordance with the present invention, include but are notlimited to, the in vivo modulation of male sexual development in thefetus, secondary sexual development at puberty and anabolic processes(muscle growth, bone density), male sex drive (libido), skin condition,hair growth and physical stamina in adults, lipid metabolism, modulationof androgen-related processes (e.g. aging, stamina, muscle tone,spermatogenesis and the like). As readily recognized by those of skillin the art, the availability of selective phyto-androgen(s) in EUextracts for the androgen receptor makes it possible, for the firsttime, to develop nutraceutics in the form of, for example, foodsupplements and natural medicines for human and animal consumptionwithout the need for special prescriptions. Such in vivo applications ofthe invention process may allow the daily modulation of variousbiological processes related to androgen action with reduced occurrenceof undesirable side effects and the like. Processes capable of beingmodulated by estrogen or via an estrogen receptor include menopause,osteoporosis and cardiovascular disease.

The ability of compounds of the invention to modulate such processes maybe evidenced in any number of ways. For example, EU extracts, in thepresence of a ligand (e.g. DHT) exert a potentiating effect on theexpression of genes under the control of androgen-response elements.

Accordingly, another aspect of the present invention is directed to acomposition comprising a part of EU or a botanical or horticulturalequivalent of EU or an extract thereof or chemical or functionalequivalents of the extract or a purified or chemical synthetic form ofone or more components of the extract wherein said composition iseffective in modulating a steroidogenic-mediated condition in a subject.

The composition of the present invention may also be referred to as aherbal composition, natural medicine, a formulation and/or a formulationor composition with medicinal or ameliorating properties. The terms“formulation” and “composition” are used herein interchangeably.

The subject formulation in the form of a part of EU or an extractthereof may be administered in any suitable form including ingestion,topical application or via vapour or aerosol means. The term “ingestion”includes administering the herb or extract via edible or liquid means.

For in vivo applications, the extract or plant parts can be incorporatedinto a pharmaceutically acceptable formulation including a carrier ordiluent for administration. Those skilled in the art will readilydetermine suitable dosage levels.

Reference herein to “suitable dosage levels” includes reference tolevels of phyto-steroidogens sufficient to provide circulatingconcentrations high enough to effect activation of steroidogenreceptor(s) or to agonize activity of a steroidogen-steroidogen receptorcomplex. Such a concentration typically falls in the range of about 1 nMup to 2 mM; with concentrations in the range of about 100 nM to 200 nMbeing preferred. Generally, however, the concentration is measured interms of w/w of dried extract or v/v of liquid extract. Exemplary massamounts are from 1 to about 80% w/w or more particularly from 5 to about50% w/w or even more preferably from 5 to about 20% w/w.

Exemplary pharmaceutically acceptable carriers include carriers suitablefor oral, intravenous, subcutaneous, intramuscular, subcutaneous, andthe like administration. Administration in the form of creams, lotions,tablets, dispersible powders, granules, syrups, elixirs, sterile aqueousor non-aqueous solutions, suspensions or emulsions, and the like, iscontemplated.

For the preparation of oral liquids, suitable carriers includeemulsions, solutions, suspensions, syrups and the like, optionallycontaining additives such as wetting agents, emulsifying and suspendingagents, sweetening, flavouring and perfuming agents, and the like.

For the preparation of fluids for parenteral administration, suitablecarriers include sterile aqueous or non-aqueous solutions, suspensionsor emulsions. Examples of non-aqueous solvents or vehicles are propyleneglycol, polyethylene glycol, vegetable oils, such as olive oil and cornoil, gelatin, and injectable organic esters such as ethyl oleate. Suchdosage forms may also contain additional ingredients such as preserving,wetting, emulsifying and dispersing agents. Formulations may besterilized, for example, by filtration through a bacteria-retainingfilter, by incorporating sterilizing agents into the compositions, byirradiating the compositions, or by heating the compositions. They canalso be manufactured as solutions in sterile water, or some othersterile injectable medium, immediately before use.

The present invention further contemplates purified or chemical forms ofone or more components of EU or its extracts. A “purified” form means acomponent which has undergone at least one purification step such asHPLC, electrophoresis, immunoprecipitation, ammonium sulphateprecipitation or high speed centrifugation.

Accordingly, another aspect of the present invention provides a purifiedor chemical synthetic molecule form of EU or a botanical orhorticultural equivalent thereof or an extract thereof which molecule iscapable of modulating an steroidogenic-mediated condition.

Purification of components in the EU extract may be readily accomplishedby any convenient means. For example, the extract may be fractionatedand fractions then tested in a cotransfection bioassay (e.g. see Ong,1999). In this assay, an EU extract or a fraction thereof is contactedwith a cell cotransfected with a steroidogen receptor expression plasmidand a luciferase-receptor plasmid containing two steroidogen responsiveelements. Luciferase activity is then a measure of steroidogenicactivity. Any other reporter molecule may be used.

Accordingly, another aspect of the present invention contemplates amethod for identifying a component of EU having steroidogenicproperties, said method comprising contacting an extract of EU or afraction of said extract with cells cotransfected with a steroidogenexpression plasmid and a genetic sequence encoding a reporter moleculecontaining an steroidogen responsive element and determining the levelof activity of the receptor molecule.

Yet another aspect of the present invention is directed to the use of EUor a botanical or horticultural equivalent thereof or an extract thereofor a chemical or functional equivalent of the extract or a purified orchemical synthetic form of one or more components of the extract in themanufacture of a medicament for the treatment of steroidogenic-mediatedconditions.

Still yet another aspect of the present invention provides a method forhormonal therapy in a subject, said method comprising administering tosaid subject an effective amount of an extract of EU or botanical orhorticultural equivalents of EU or chemical or functional equivalents ofthe extract or a purified or chemical synthetic form of one or morecomponents of the extract for a time and under conditions to modulatesteroidogenic activity.

In one embodiment, the steroidogenic activity is androgenic activity.This is important for modulating male sexual development, sexualfunction and infertility amongst other conditions.

In another embodiment, the steroidogenic activity is estrogen activity.The latter is important for the treatment of menopause, osteoporosis andcardiovascular disease amongst other conditions.

The present invention is further described by the following non-limitingExamples.

EXAMPLE 1 Extraction and Formulation

One kg of bark of the EU plant, or other plants as described in Example2 below, was ground into powder with a grinding machine. The groundplant bark was placed in a vessel and the active compounds withsteroidogenic activities were extracted with 10 litres of 100% ethanolby soaking the ground bark for 1 week, to allow the active compounds todissolve into the solvent system. The liquid phase was then separatedfrom the solid phase by filtration with a Whatman-1 filter paper.Samples prepared in this manner are hereinafter referred to as“ethanolic extracts”.

In some instances, a portion of the ethanolic extract was then furtherpurified by addition of distilled water to 20% v/v. Material thatprecipitates as a result was removed by filtration with Whatman-1 filterpaper. The resulting liquid fractions are hereinafter referred to as“hydroethanolic extracts”.

EXAMPLE 2 Identification of Herbal Extracts Which Activate AndrogenReceptor

As part of an ongoing bioprospective search for ethanobotanical herbswhich contain novel phyto-androgens that act through the androgenreceptor, a multitude of extracts, prepared as in Example 1 frommedicinal herbs identified by pharmacognosy, were screened utilizing acotransfection bioassay similar to that described by Ong, 1999

Candidate herbal extracts were initially tested in HeLa cellscotransfected with an androgen receptor expression plasmid and aluciferase-receptor plasmid containing two androgen responsive elements(ARE). In this assay the androgenic activity of any added compound canbe accurately measured.

Thus, HeLa cells were cotransfected with an androgen receptor (AR)expression plasmid, a plasmid for expression of β-galactosidase (β-gal)and a luciferase reporter plasmid, ARE-Tata-luc (Jenster 1997) and thenincubated with ethanol as a solvent control, or with the herbal extractsindicated in Table 1. Herbal extracts were added to the cells six hoursafter transfection. Thirty-six hours later, the cells were harvested andcell extracts assayed for luciferase activity.

The AR expression and reporter gene vectors were transfected into HeLacells, a human cell line exhibiting minimal endogenous AR expression,using the lipofection technique. The DNA mix contained an AR expressionvector, a reporter gene vector (ARE-Tata-Luc) and a βGal vectors, thelast for purposes of normalizing results for the transfectionefficiency. The AR expression vector comprises a cDNA encoding AR andexpressed from a SV40 promoter and a rabbit β-globin polyadenylationsignal, all cloned in a pBR328 vector. The β-gal vector comprises abacterial lacZ gene under control of an SV40 promoter-enhancer. The DNAmix was preincubated for 45 min at room temperature with lipofectamine(Promega, Madison, Wis.) in a 400 μl serum-free medium. The DNA-liposomecomplexes (total volume of 200 μl) were overlaid onto 50-60% confluentHeLa cells growing in 24 well plate. Transfection was continued for 5hours before removal of the transfection mix and the replacement withgrowth medium containing 10% charcoal-stripped fetal calf serum, andindicated amounts of androgens and/or extract. After 40 to 48 h ofincubation, the cells were rinsed twice with PBS, and lysed with 400 μlof reporter lysis buffer (Promega). Cells were scraped from the wellsand after one freeze thaw cycle, the cell lysate was cleared bycentrifugation at 12,000×g for 10 min. Cell lysates (100 μl) were addedto 20 μl of luciferase substrate and luciferase activity measured with aluminometer. Transfection efficiency was assessed by βGal activity andluciferase activity normalized to the protein content of the celllysates.

Total protein in the supernatant was quantified with bovine serumalbumin as the standard.

Table 1a shows that an ethanolic EU extract displayed significantandrogenic activity increasing reporter gene activity, 41-fold (7.8% ofthe activity observed with 1 nM DHT), compared to controls not exposedto DHT or herbal extract. In contrast, two species of ginseng, Panaxquinquefolius and Panax ginseng did not induce significant androgenreceptor activity, giving only of 1.7-fold (0.3% of 1 nM DHT) and1.9-fold (0.4% of 1 nM DHT) increases respectively. Similarly, JamuTongkat Ali (A Malayan aphrodisiac herbal composition comprising ofEngenia aromatica, Trigonella graecum, Zingiber officinale, pipernigrum, Cinnamomum sarivum, Amomum kepulaga, Cinnamomum zeylanicum,Eurycoma longifolioa) demonstrated little androgenic activity with aninduction value of only 1.2-fold (0.2% of 1 nM DHT).

Table 1b shows that only an ethanolic EU extract was able to potentiateandrogen activity, by increasing DHT activity to 187% of that observedwith DHT alone. In comparison, none of the other herbal extracts, —SB(Evodia rutaecarpa), SE (Syzygium aromaticum), SY (aconitumcarmichaeli), SZ (Alpinia oxyphylla) were able to potentiate the effectof DHT on the AR.

Table 1c shows that an ethanolic EU extract did not activate the closelyrelated progesterone and glucocorticoid receptors, showing only 1.3 and2.2-fold activity, respectively, compared to 41-fold activity observedwith the androgen receptor above (Table 1a). Similarly, in the presenceof their respective ligands, an ethanolic EU extract did not potentiatePR and GR activity. The presence of an ethanolic EU extract and cognateligands induced only 64 and 99% of the activity observed with thecognate ligands alone, when tested with the progesterone andglucocorticoid receptors, respectively. Thus, the stimulatory activityof an ethanolic EU extract on the androgen receptor was not observedwith the glucocorticoid or progesterone receptors.

EXAMPLE 3 Transactivation Studies

To further characterize the androgenic activity of the EU, the extractalone (either ethanolic EU extract or hydroethanolic EU extract asindicated accordingly), and in combination with testosterone and DHT,were tested for their transactivation properties.

FIG. 1 shows the dose-dependent responses of the five differenttreatments:

-   -   FIG. 1 a—different concentrations of an ethanolic EU extract    -   FIG. 1 b—different concentrations of a hydroethanolic EU extract        (after 20% water precipitation of insoluble compounds).    -   FIG. 1 c—increasing doses of testosterone in the presence of        50ng/ml of an ethanolic EU extract    -   FIG. 1 d—increasing doses of DHT in the presence of 50ng/ml of        an ethanolic EU extract    -   FIG. 1 e—increasing doses of an ethanolic EU extract in the        presence of 1 nM DHT.

Experiments in HeLa cells were conducted as described in Example 2. Asshown in FIG. 1 a, ethanolic EU extract alone demonstrates intrinsicandrogenic activity by increasing Luc activity to a maximal 1.87-fold ina dose-dependent manner. After 20% water precipitation of the ethanolicEU extract (=hydroethanolic EU extract), some phyto-metabolites that arecell inhibitory were removed, resulting in a more consistentdose-response curve with a maximal induction of over 6-fold (FIG. 1 b).This shows that ethanolic extraction increases the concentration of theandrogenic compound(s), and that further precipitation with water(ethanol/water, 80:20) will further enhance the steroidogenic activitiesof the ethanolic extract.

Most strikingly, an ethanolic EU extract can synergise and augment theeffects of physiological androgens. In FIG. 1 c, testosterone, at a doseof >1 nM, was able to induce maximal androgen receptor activity to about100-fold higher than replicates not exposed to androgen. Addition of anethanolic EU extract raised this maximum further to over 200-fold, anincrease of about 100% compared to testosterone alone. This novelsynergistic action on maximal androgen receptor activity was alsoevident with DHT. The presence of an ethanolic EU extract shiftedupwards the maximum of the DHT dose-response curve by 40-60% (FIG. 1 d).To further confirm this unprecedented synergistic bioactivity,increasing concentration of an ethanolic EU extract was added to DHT. Asshown in FIG. 1 e, an ethanolic EU extract dose-dependently raisedandrogen receptor activity above that observed with a saturating dose ofDHT (1 nM).

EXAMPLE 4 Competitive Radioligand Displacement Bioassays

To demonstrate that the transactivation of the androgen receptor is dueto the specific binding of the phyto-androgen(s) contained in anethanolic EU extract, competitive radioligand displacement assays werecarried out (Wilson, 1976). In this assay, androgen receptors expressedin COS-7 cells were simultaneously exposed to increasing concentrationsof an ethanolic EU extract and a limiting amount (3 nM) of tritiatedtestosterone. The ability of an ethanolic EU extract to compete withradiolabelled testosterone for androgen receptor binding is compared toa strong androgen (DHT) and a non-ligand, cortisol. Thus, DHT andcortisol are positive and negative controls, respectively. The treatedcells were harvested and the amount of tritiated testosterone bound toandrogen receptor was measured by scintillation counting. Specificbinding is expressed as percent tritium bound to androgen receptor,where 100% was the amount of specific tritiated-testosterone bound inthe absence of competing cold ligand minus background (non-specificbinding to substrate and proteins).

As shown in FIG. 2, an ethanolic EU extract competes well with tritiatedtestosterone, showing a graduated displacement of tritiated testosteroneas the concentration of an ethanolic EU extract was increased, attaining62.6% displacement at cf. 10. In contrast, the non-ligand cortisol doesnot displace tritiated testosterone at concentrations ranging from 0.3to 10 nM. The androgen DHT also displaces tritiated testosterone in adose-responsive pattern, attaining 92.4% displacement at 10 nM DHT. Thisdemonstrates that the displacement of tritiated testosterone fromandrogen receptor is restricted to true androgen receptor ligands and anethanolic EU extract that contain phyto-androgens that bindsspecifically to the ligand binding pocket of the androgen receptor LBD.

EXAMPLE 5 Estrogenic Activity in Herbal Extracts

The ethanolic EU extract was tested for estrogenic activity in HeLacells cotransfected with an estrogen receptor expression plasmid and aluciferase-receptor plasmid containing estrogen responsive elements(ERE). In this assay the estroegnic activity of any added compound canbe accurately measured. The results are shown in FIGS. 3 to 5. FIG. 3shows that an ethanolic EU extract has estrogenic activity on its own asit can increase the response of the reporter gene to a maximum of1.6-fold, compared to 4-6 fold for estradiol. FIG. 4 shows that the sameextract can potentiate the effect of estradiol by doubling the activityof the reporter gene compared to estradiol alone. This synergisticactivity of an ethanolic EU extract on estrogenic activity was alsoobserved when increasing doses of ethanolic EU extract were co-incubatedwith 0.1 nM estradiol (FIG. 5).

The ability of the herbal extract to modulate estrogenic activityprovides a method for conducting hormonal therapy in hypoestrogenicstates such as menopause, osteoporosis and cardiovascular disease.

EXAMPLE 6 In-Vivo Androgen Modulatory Activity in Animal Studies

To determine the androgenic effect of an ethanolic EU extract inanimals, the growth of the ventral prostate gland, an androgen-dependenttissue, in immature Wistar rats was measured. These immature rats havelow levels of testosterone and consequently underdeveloped prostatetissue. In this model, exogenous androgen adminstration can be directlycorrelated with the growth of the ventral prostate. Thus, adose-dependent increase in prostate weight (per unit body weight) can beobserved when testosterone was administered intramuscularly (FIG. 6 a).Administration of an ethanolic EU extract alone at doses from 1 mg to 10mg also resulted in dose-dependent increments in prostatic weight (FIG.6 b).

Interestingly, the total androgenic effect of an ethanolic EU extractand testosterone was higher than that observed with high doses oftestosterone alone. This novel ability of an ethanolic EU extract toincrease prostatic growth to a level above that observed with maximaltestosterone levels, was seen clearly in an experiment wherein animalswere administered a high dose of testosterone (5000 ug) and also orallyfed 50 mg of an ethanolic EU extract (FIG. 6 c). These experimentsindicate that an ethanolic EU extract was able to exert a synergisticeffect on androgen action when administered orally, consistent with thesynergistic effect observed in the cell studies shown in FIG. 1.

EXAMPLE 7 Solid Phase Separation of Compounds Exhibiting SteroidogenicActivity

In an effort to further define the composition of the bioactivecompound(s) in an ethanolic EU extract with steroidogenic activity, wefurther separated the compounds using a solid phase extractionprocedure. A Diol matrix was loaded into a glass column and an ethanolicEU extract was dry packed on top of the Diol matrix. Diol matrix is apolar sorbent that can exhibit both polar and weak non-polarinteractions, the surface silanol groups are available for additionalsecondary polar interactions. Diol matrix consists of sphericalparticles of silica with Diol functions (—(OH)₂) at the end ofhydrocarbon chains (—CH₂—). The hydrocarbon chains are attached to thesilica particles by alkyl ether spacers.

The ethanolic EU extract was then sequentially eluted into individualfractions using the following solvents of increasing polarity in thefollowing order: hexane 100%, hexane:DCM (1:1) 100%, DCM 100% andmethanol 100%. The fractions were dried down in a rotary evaporator at37° C. and then resuspended in ethanol for AR bioassay. It was observedthat synergistic androgenic activity (with MB) was present in all thefractions, but with the most activity observed in the methanol 100%fraction. (FIG. 7 a). Since methanol is a polar organic solvent, thisindicates that one of the most androgenic compound(s) is an organicmolecule of mixed polarity. It also indicates that the androgenicactivity is likely to be the result of several compounds, as allfractions exhibited androgenic activity, albeit to a lesser degree thanthe methanol 100% fraction.

To further define the androgenic compounds, we used solvents of furtherdefined polarity in the Diol matrix columns and studied theirsynergistic effects with the physiological androgen, DHT (FIG. 7 b). Weused the solvents DCM, ethyl acetate and ethanol (EtOH) as they coverthe range of polarity shown to have the greatest activity in FIG. 7 a.Synergistic androgenic activity eluted with the first fraction ofethanol (EtOH I), indicating that one class of active compound(s) is ofrelatively high polarity.

EXAMPLE 8 Thin Layer Chromatography (TLC) Separation of SteroidogenicCompounds

An ethanolic EU extract was loaded onto a Sephadex LH-20 column andeluted with n-butanol. Sephadex LH-20 matrix is a hydroxypropylated,cross-linked dextran matrix having an exclusion limit for peptides of 4kilodaltons and an exclusion limit for small organic molecules of 5kilodaltons and has dual lipophilic and hydrophilic properties. It is acrosslinked dextran gel that derives its lipophilic character from theisopropyl groups (—CH₂—CH—CH₂—) present. The hydrophilicity is due tothe numerous hydroxyl functions present (—OH). Dextran (Dex) is ageneral term referring to linear highly polymeric carbohydrates arisingas metabolic products of the bacterium Leuconostoc mesenteroides growingon a cane sugar substrate. Sephadex LH-20 matrix consists of Dexcross-linked via glycerol ether bridges.

The butanol eluate was further fractionated by two-dimensional thinlayer chromatography (TLC). 100 u of the butanol eluate was spottedband-wise on a TLC Silica Gel-60 glass plate at the baseline. The TLCplate was then developed in a chamber with 10 ml of the first solventsystem, 100% dichloromethane. After 20 mins, the TLC plate was turned 90degrees with respect to the first baseline and a second solvent system,100% hexane was used to develop the 2-D TLC plate for 10 min. After thesecondary exposure to 100% hexane, the TLC plate was air-dried at roomtemperature.

The TLC plate was then visualized under UV illumination. Multiplefluorescent bands of different colors such as blue and yellow were seenspread between baseline (‘B0’, R_(f)=0) to a point ‘B1’, having an R_(f)of 0.4. The most prominent bands visible under UV were found at ‘B0’ and‘B 1’.

Subsequently, the TLC plate was chemically reacted with avanillin-sulphuric acid spray and the plate baked at 90° C. for 30 min.This time, various hues of violet-blue bands and brown bands were seendistributed between ‘B0’ to ‘end’. The most prominent of these bandswere marked as ‘B2’, ‘B3’, ‘B4’ and ‘end’ with corresponding R_(f)values are 0.47,0.62, 0.71 and 1 respectively.

A composition of the invention is preferably one that lacks at least oneof the components B0, B1, B2, B3, B4 and “end”, as these representcompounds that are not bioactive according to the present invention.

The bands and regions between those bands that were visualized by UV andvanillin-sulphuric acid reagent (i.e. the ‘interbands’) were thenscraped off as silica gel-60 powders from another intact duplicate TLCplate based on the R_(f) values. The original TLC plate used for thevanillin-sulphuric acid reaction cannot be re-used for further analysesas the vanillin-sulphuric acid chemical reaction is a destructiveprocess.

After the silica gel-60 powders containing the TLC bands and‘interbands’ were scraped off, they were then individually incubated at37° C. for 12 hours in 10 ml aliquot of 100% ethanol in separatecentrifuge tubes to elute the EU compounds into solution for thebioassays.

FIG. 8 shows that a largely hydrophobic n-butanolic fraction, obtainedby passing an ethanolic EU extract through a Sephadex LH-20 column, canbe further differentiated into bands of phytocompounds that possesssubtle differences in hydrophilicity based on hydrophilic interactionwith the silanol groups of the silica gel stationary phase of a thinlayer chromatography (TLC) plate. This shows that the AR agonistactivity of an ethanolic EU extract is found in more than one singlecompound. Also, the bioactive compounds possess mixed polarities basedon the Sephadex LH-20 and TLC chromatographic separations. A(non-limiting) example of a mixed polarity compound would be a saponin(with a steroidal or triterpenoid aglycone coupled to sugar moieties);or a flavonoid with conjugated hydrophobic aromatic ring structurescarrying entities such as hydrophilic hydroxyl or amide side-groups; oran Okadaic acid-type conjugated ring structure system with chargedside-groups such as (but not restricted to) methyl, hydroxyl or amidegroups; or fatty-acids derivatives and/or conjugates such aslipopolysaccharides (LPS).

EXAMPLE 9 High Performance Liquid Chromatography (HPLC) Separation ofSteroidogenic Compounds

10 mg of dried EU ethanolic extract was dissolved in 2 ml ofacetonitrile and applied to a C-18 reverse phase HPLC column. Thechromatographic mobile phase consists of acetonitrile and water in thefollowing percentages: Percentage acetonitrile to Flow type Duration(min) water in the mobile phase Isocratic  0-15  10% acetonitrileGradient 15-40  10% to 50% acetonitrile Gradient 40-60  50% to 100%acetonitrile Isocratic  60-100 100% acetonitrile Gradient 100-120 100%to 10% acetonitrile

Fractions F1, F2 and F3 are broad time-based fractions. F1 was collectedfrom 40 min to 60 min, F2 was collected from 60 min to 80 min and F3 wascollected from 80 min to 120 min.

FIG. 9 indicates that the androgenic activity of an ethanolic EU extractcan be separated into different fractions with varying degree ofagonistic and androgen-boosting effects. This shows that theandrogenic/booster bioactivities of an ethanolic EU extract are mediatedby different classes of phytocompounds which can be separated on areverse phase C-18 matrix. The hydrophobic stationary reverse phase(C-18) matrix was able to interact with and resolve the ethanolic EUextract into three fractions with differing bioactivatingcharacteristics. This demonstrates that the active molecules responsiblefor androgenic and androgen-boosting bioactivity are of differentpolarities with at least some hydrophobic moieties and/or differenttype(s) of chemical structures. In other words, an ethanolic EU extractcontains more than one compound having biological action on the AR.

The elution profiles of three experiments are superimposed in FIG. 10.Two major peaks can be identified in fraction F2, having retentionstimes of 61.6 minutes and 64 minutes, respectively.

EXAMPLE 10 Combination Chromatographic Separation of Active Compounds

The chromatographic approaches to separating steroidogenic compoundsaccording to the invention, especially those compounds having asynergistic action with steroids, preferably with androgens andestrogens, can be applied in combination to obtain more purifiedfractions. One preferred combination is to obtain an alcoholic eluatefrom a diol column and apply that eluate to a reverse phase column. Inan example of this approach, an ethanol, hexane or DCM extract of EU isapplied to a diol column by dry packing and then the diol column iseluted with DCM followed by ethyl acetate then by ethanol. The ethanoleluate is collected and applied to a reverse phase C-18 column (eitherdirectly or by dry packing). The reverse phase column is eluted with anisocratic mobile phase of methanol:water (1:1 by volume) followed by100% methanol, then followed by 100% DCM. The DCM eluate is collected asthe active fraction. Three column volumes of mobile phase are used foreach elution step for both columns.

EXAMPLE 11 Effective Dosage in Humans

Those skilled in the art, will recognize that the examples describedabove allow a prediction of the dose that will be effective in humans.The in vitro cell culture experiments indicate that synergistic activitycan be observed at doses ranging from 0.05 ng/ml to 500 ng/ml of theethanolic EU extract or hydroethanolic EU extract. Since the human bloodvolume is 5000 mls, it is anticipated that assuming 100% absorption, adose of 0.25 μg to 2.5 mg of dried ethanolic EU extract orhydroethanolic EU extract can be effective. Animal studies indicate that1 mg/200 g to 50 mg/200 g body weight of dried ethanolic EU extractadministered orally exhibits androgenic effect. Assuming that theaverage male weighs 70 kg, the effective oral dose per weight basiswould range from 350 mg to 17.5 g of dried ethanolic EU extract. Such adose would be administered at least once every other day, preferablyone, two, three or four times per day.

A protocol for clinical trial of the extract of the invention has beensubmitted. In the trial, dried EU extract of the invention will beformulated into capsules for oral administration to subjects in dosesfrom 500 to 3000 mg, administered two or three times per day.

Patients: 21 to 30 males with primary hypogonadism, aged 20 to 60 years,with serum testosterone levels below the normal range (testosterone <5ng/ml) and raised serum gonadotrophins (FSH and LH >7 IU/L) but who areotherwise healthy.

Primary Endpoint Measures:

The main outcome measure will be significant suppression ofgonadotrophins (FSH and LH) to 60% of baseline levels; return to normallevels is desired. Secondary outcome measures will beandrogenic-regulated changes in SHBG, HDL-cholesterol, hemoglobin andprostate specific antigen levels. Return of these physiologic parametersto normal levels is desired.

Other outcome measures for subsequent trials (applicable for both menand women) include psychometric indices of sexual function/libido andmood/feelings; dual energy X-ray absorptiometry (DEXA) and/or magneticresonance imaging (MRI) measurements of body proportions of fat, leanbody mass, muscle bulk distribution and bone mineral density;dermatologic evaluation for hydration, pigmentation and sebum productionin skin. FSH and LH suppression are also applicable for women. TABLE 1¹TABLE 1a Androgenic activity of herbal extracts Androgenic (%)percentage activity (fold of 1 nM DHT Herbal extract induction) activityEthanolic EU extract 41 7.8 P. ginseng 1.9 0.4 P. quinquefolius 1.7 0.3Jamu Tongkat Ali 1.2 0.2 TABLE 1b Androgenic activity of herbal extractsin the presence of DHT (1 nM). Herbal extract + DHT alone DHT(Androgenic (Androgenic Ratio (%) activity fold activity fold (Herbalextract + Herb induction) induction) DHT/DHT alone) SB 29 183 15.8 SE162 183 88.5 SY 175 183 95.6 SZ 187 183 102 Ethanolic 403  215² 187 EUextract TABLE 1c Absence of transactivation activity of ethanolic EUextract with progesterone and glucocorticoid receptors Luc activityRatio of Luc activity (fold as percentage (%) Treatment³ induction) of 1nM ligand Ethanolic EU extract + 1.3 1.9 progesterone receptor EthanolicEU extract + 43 64 progesterone + progesterone receptor Progesterone +67 100 progesterone receptor Ethanolic EU extract + 2.2 1.1glucocortcoid receptor Ethanolic EU extract + 192 99 cortisol +glucocortcoid receptor Cortisol + 194 100 glucocortcoid receptor¹Wild type androgen receptor was transiently expressed in HeLa cells andexposed to 1 nM DHT or 50 ng/ml herbal extracts (Table 1a). HeLa cellswere exposed to 1 nM DHT in the presence of different herbal extracts(Table 1b).# Ethanolic EU extracts tested for progesterone and glucocortcoidactivity (Table 1c). Transactivation activity was measured with anandrogen-regulated reporter gene, ARE-Tata-Luc and expressed as foldincrease in luciferase activity compared with cells not exposed toandrogen or herbal extract. # Progesterone or glucocorticoid receptorswere utilized when testing for progestogenic or glucocorticoidactivities respectively. Each data point is the mean of triplicatesamples.²Difference in DHT androgenic activity compared to that of other herbsis because the ethanolic EU extract assay was performed on a differentday.³Fold increase in luciferase reporter gene activity coupled toappropriate hormone response element, compared to wells not exposed tohormone or herbal extract. Cells were exposed to 1 nM of eitherprogesterone or cortisol.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

Various articles of the scientific and patent literature are citedthroughout this paper. Each such article is hereby incorporated byreference in its entirety and for all purposes by such citation.BIBLIOGRAPHY

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1. A composition comprising a preparation of Eucommia ulmoides or of ahorticultural equivalent of Eucommia ulmoides prepared by alcohol orwater extraction thereof and chromatography a) over a hydroxypropylated,cross-linked dextran matrix having an exclusion limit for peptides of 4kilodaltons and an exclusion limit for small organic molecules of 5kilodaltons with elution using n-butanol or b) over a reverse phasematrix with elution by an aqueous buffer and acetonitrile gradient or c)over a diol matrix with elution using at least one organic solvent; thatlacks fluorescent components exhibiting R_(f) values of 0 and 0.4 andalso lacks vanillin-sulphuric acid reactive components exhibiting R_(f)values 0.47,0.62, 0.71 and I when the composition is separated by twodimensional thin layer chromatography on silica gel and eluted in thefirst dimension with dichloromethane and in the second dimension withhexane.
 2. A method of modulating a steroid-mediated physiologicalcondition in a subject comprising administering to said subject anamount of a formulation of the composition of claim 1 that is effectivefor modulating the steroid-mediated physiological condition in thesubject.
 3. The method according to claim 2 wherein the preparation isadministered to a human, a primate or a livestock animal.
 4. The methodaccording to claim 2 wherein the steroid-mediated physiologicalcondition is mediated by an androgen or by androgen receptor.
 5. Themethod according to claim 2, wherein the steroid-mediated physiologicalcondition is male sexual development, secondary sexual development,anabolic processes, male sex drive, skin condition, hair growth,physical stamina or lipid metabolism.
 6. The method according to claim 2wherein the steroid-mediated physiological condition is mediated byestrogen or estrogen receptor.
 7. The method according to claim 2,wherein the steroid-mediated condition is menopause, osteoporosis,cardiovascular disease or other estrogen-related disease or process. 8.The method according to claim 2 wherein the formulation comprises apurified or chemically synthesized form of a component of an alcohol orwater preparation of Eucommia ulmoides.
 9. A method for producing asteroidogenic preparation from Eucommia ulmoides comprising: i)optionally macerating Eucommia ulmoides plants or at least one partthereof; ii) obtaining a solvent preparation by soaking the Eucommiaulmoides plants or parts thereof in a solvent system; iii) separatingthe solvent preparation from insoluble materials; iv) obtaining anpreparation residue by drying the solvent preparation; v) dry packingthe preparation residue on a diol column; and vi) obtaining an eluatecomprising steroidogenic compounds from the column by eluting using atleast one of hexane, hexane:dichloromethane 1:1, dichloromethane, ethylacetate, or a C₁ to C₆ alkane alcohol; thereby obtaining a steroidogenicpreparation.
 10. The method of claim 9, further comprising: iiia)precipitating undesired compounds by adding water in an amount of up to20% v/v; and iiib) separating the solvent preparation from theprecipitate; prior to step iv).
 11. The method of claim 10, wherein theC₁ to C₆ alkane alcohol is ethanol or methanol.
 12. The method of claim10, wherein the diol column is eluted with a first solvent that ishexane, hexane:dichloromethane 1:1, dichloromethane, ethyl acetate, or aC₁ to C₆ alkane alcohol and then is eluted with a different solvent thatis hexane, hexane:dichloromethane 1:1, dichloromethane, ethyl acetate,or a C₁ to C₆ alkane alcohol.
 13. A method for producing a steroidogenicpreparation from Eucommia ulmoides comprising: i) optionally maceratingEucommia ulmoides plants or at least one part thereof; ii) obtaining asolvent preparation by soaking the Eucommia ulmoides plants or partsthereof in a solvent system; iii) separating the solvent preparationfrom insoluble materials; iv) applying the preparation to a reversephase chromatography column and eluting the steroidogenic compounds witha mobile phase comprising water and acetonitrile in an amount thatvaries over time from 10% to 100% acetonitrile; thereby obtaining asteroidogenic preparation.
 14. The method of claim 13, furthercomprising: iiia) precipitating undesired compounds from the solventpreparation by adding water in an amount of up to 20% v/v; and iiib)separating the solvent preparation from the precipitate; prior to stepiv).
 15. A method for producing a steroidogenic preparation fromEucommia ulmoides comprising: i) optionally macerating Eucommia ulmoidesplants or at least one part thereof; ii) obtaining a solvent preparationby soaking the Eucommia ulmoides plants or parts thereof in a solventsystem; iii) separating the solvent preparation from insolublematerials; iv) applying the preparation to a chromatography matrixcomprising dextran crosslinked by an alkyl ether and eluting thesteroidogenic compounds with a C₁-C₆ alcohol; thereby obtaining asteroidogenic preparation.
 16. The method of claim 15, furthercomprising iiia) precipitating undesired compounds from the solventpreparation by adding water in an amount of up to 20% v/v; and iiib)separating the solvent preparation from the precipitate; prior to stepiv).
 17. A composition comprising the preparation produced by the methodof any one of claims 9, 13 or
 15. 18. A composition comprising a portionof the preparation produced by the method of claim 13, that elutes atany one of: i) 100% acetonitile; or ii) a gradient of 50% to 100%acetonitrile.
 19. A composition comprising a combination of the eluatesobtained by the method of claim
 12. 20. An article of manufacturing acomposition according to claim 1 and written material that providesinstructions or urges the use of the composition to modulate, aphysiological condition or response mediated by a steroid.